Anti-idiotypic antibody which induces an immune response against a glycosphingolipid and use thereof

ABSTRACT

The present invention provides an anti-idiotypic monoclonal antibody which specifically induces an immune response against a glycosphingolipid. Additionally, this invention provides a method of producing the anti-idiotypic monoclonal antibody. Finally, this invention provides a composition of matter comprising an effective amount of a cytokine and a melanoma ganglio-side-specific antibody attached to a carrier.

This application is a divisional of U.S. Ser. No. 08/447,412, filed May 23, 1995 now, U.S. Pat. No. 6,432,402, which is a continuation of U.S. Ser. No. 08/218,316, filed Mar. 25, 1994 now abandoned; which is a continuation of U.S. Ser. No. 07/776,266 filed Jan. 27, 1992; now abandoned; which is a continuation Application of PCT International Application No. PCT/US90/03061, filed May 25, 1990, which is a continuation in part and claims priority of U.S. application Ser. No. 07/357,037, filed May 25, 1989, now abandoned.

BACKGROUND OF THE INVENTION

R24, an IgG3 mouse monoclonal antibody (mAb) raised against a human melanoma, recognizes the GD₃ ganglioside. The GD₃ ganglioside is abundantly expressed on most melanomas, however, expression of the GD₃ ganglioside on normal tissue is selective and occurs at low concentrations (1). Gangliosides are a subset of glycosphingolipids which in turn are a subset of glycolipids. Glycolipids, as their name implies, are sugar-containing lipids.

R24 comprises a known mixture of three variant antibody species (FIG. 11) designated V1-R24, V2-24, and R24. The V1-R24 and V2-R24 variants comprise one and two irrelevant antibody light chains, respectively; in contrast. R24 comprises relevant light chains. Generally, light and heavy antibody chains in combination effect specific antibody-antigen binding. However, antibodies comprising irrelevant antibody light chains, unlike antibodies comprising relevant antibody light chains, do not specifically bind an antigen.

In order to avoid confusion, the R24 variant from hereinafter will be referred to as “R24” and R24 comprising the three variant species will be referred to as the “R24 composition.” Each variant exhibits differences which imply that the GD₃-binding region (also termed “paratope”) of V1-R24, V2-R24, and R24 are altered.

Anti-idiotypic monoclonal antibodies which mimic and possess the internal image of a saccharidic epitope have been developed (2). The saccharidic epitope is a tumor associated globoside, i.e. a glycolipid. However, these anti-idiotypic (monoclonal antibodies do not specifically mimic a glycosphingolipid, a ganglioside, or more particularly the GD₃ ganglioside. Additionally, these anti-idiotypic monoclonal antibodies are not directed against R24 or any antibodies which specifically bind a glycosphingolipid or a ganglioside, let alone the GD₃ ganglioside.

Mice have been primed against capsular antigens of pathogenic bacteria by administration of monoclonal anti-idiotypic antibodies which mimic the polysaccharide capsule of E. coli (a gram negative bacteria) (3). It is generally known that lipopolysaccharides (LPS) is a major component of the outer membrane of gram negative bacteria and is released when bacteria are lysed. LPS consists of a heteropolysaccharide chain linked covalently to a glycolipid. It is important to point out that although these monoclonal anti-idiotypic antibodies were used as a means to prime a subject against a LPS, there is no suggestion of generating monoclonal anti-idiotypic antibodies against R24 or any antibodies which would specifically bind glycosphingolipids or gangliosides, in particular the GD₃ ganglioside.

The GD₃ ganglioside is poorly immunogenic (1). Accordingly, the GD₃ ganglioside is an appealing target for cancer immunotherapy because of the commercial utility of designing molecules which would structurally mimic GD₃ and possess dramatically significant immunogenic properties.

SUMMARY OF THE INVENTION

The present invention also provides an anti-idiotypic monoclonal antibody which specifically induces an immune response against a glycosphingolipid and more specifically against a ganglioside. Additionally, this invention provides a composition of matter comprising an effective amount of a cytokine and a melanoma ganglioside-specific antibody attached to a carrier. Finally, this invention provides a method of generating the previously-identified anti-idiotypic antibody which comprises: (a) preparing an antibody; (b) purifying the antibody; (c) attaching the antibody onto a carrier so as to produce an immunogen; (d) combining the immunogen with a cytokine so as to produce a novel immunogen-cytokine combination; and (e) injecting the novel immunogen-cytokine combination into a mammal thereby producing the anti-idiotypic antibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Inhibition of R24 binding to GD₃ by BEC2. Dilutions of BEC2 were incubated with 5 μg/ml of biotinylated R24 for 1 hour. The mixture was then plated into wells coated with melanoma gangliosides containing GD₃. Binding of R24 was detected by adding alkaline phosphatase-conjugated avidin for 15 minutes. After washing, substrate (p-nitrophenylphosphate) was added and the absorbance at 410 nm was measured.

FIG. 2: Anti-GD₃ reactivity induced in rabbits by immunization with BEC2. Rabbits were inoculated subcutaneously with 100 μl of BEC2 in complete Freund's adjuvant. Subsequent booster immunizations were administered either subcutaneously in incomplete Freund's adjuvant (Days 17 and 31) or intramuscularly without adjuvant (Days 57 and 85). To detect anti-GD₃ rabbit antibodies by ELISA, 96-well plates were coated with purified melanoma GD₃ and blocked with 5% non-fat milk. Dilutions of rabbit serum were added for 1 hour. After washing, alkaline phosphatase-conjugated anti-rabbit IgG was added. Binding was visualized by adding substrate (p-nitrophenyl-phosphate) and measuring the absorbance at 410 nm.

FIG. 3: Anti-GD₃ reactivity induced in rabbits by immunization with BEC2. Rabbits were inoculated subcutaneously with 100 μl of BEC2 in complete Freund's adjuvant. Subsequent booster immunizations were administered either subcutaneously in incomplete Freund's adjuvant (Days 17 and 31) or intramuscularly without adjuvant (Days 57 and 85). To detect anti-GD₃ rabbit antibodies by ELISA, 96-well plates were coated with purified melanoma GD₃ and blocked with 5% non-fat milk. Dilutions of rabbit serum were added for 1 hour. After washing, alkaline phosphatase-conjugated anti-rabbit IgM was added. Binding was visualized by adding substrate (p-nitrophenyl-phosphate) and measuring the absorbance at 410 nm.

FIG. 4: Inhibition by BEC2 or R24 binding to GD₃. Data shows significant inhibition of R24 binding to GD₃ by BEC2, this is consistent with BEC2 being an Ab 2b anti-idiotypic mAb.

FIG. 5: Anti-GD₃ IgG of Rabbit 521 by RIA. Rabbit 521 developed IgG against GD₃ after being immunized with BEC2 mixed with Freund's complete adjuvant. The response was measured by RIA.

FIG. 6: Anti-GD₃ IgM of Rabbit 543 by ELISA. Rabbit 543 developed IgM after being immunized with BEC2 mixed with Freund's complete adjuvant. The response was measured by ELISA.

FIG. 7: Anti-GD₃ IgM of Rabbit 545 by ELISA. Rabbit 545 developed IgM after being immunized with BEC2 mixed with Freund's complete adjuvant. The response was measured by ELISA.

FIG. 8: Anti-GD₃ antibodies induced by BEC2 do not cross-react with other gangliosides. 1 μg of various purified gangliosides were applied to strips of nitrocellulose filters and allowed to dry. The strips were blocked in 5% non-fat milk and washed. Strips were incubated in rabbit serum (diluted 1:10) overnight. After washing, peroxidase-conjugated anti-rabbit IgM was added for 2 hours. The strips were again washed and binding was visualized by adding 4-chloro-1-naphthol substrate with H₂O₂.

FIG. 9: Chromatogram showing that most proteins eluted at a molecular weight of approximately 120 kD which is consistent with IgG (145-150 kD) but not consistent with IgM (900 kD).

FIG. 10: Anti-GD₃ reactivity of NH₄So₄ precipitate from Rabbit 543. Graph showing the anti-GD₃ reactivity of each fraction. It is clear that only the 120 kD fraction is bound to GD₃.

FIG. 11: R24 comprises a known mixture of three variant antibody species designated V1-R24, V2-R24, and R24. The V1-R24 and V2-R24 variants comprise one and two irrelevant antibody light chains, respectively; in contrast, R24 comprises relevant light chains.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an anti-idiotypic antibody, such as a polyclonal but preferably a monoclonal antibody, which induces an immune response against a glycosphingolipid. As used herein glycosphingolipids and gangliosides are either naturally-occurring or synthetic molecules.

In one instance the glycosphingolipid is a ganglioside, e.g. GD₃, GD2, GM2, and GM3. GD₃ is preferred.

Generally, an antibody comprises two molecules, each molecule having two different polypeptides, the shorter of which functions as the light chains of the antibody and the longer of which polypeptides function as the heavy chains of the antibody. However, as used herein, antibody is given a functional definition, i.e. any molecule, whether naturally-occurring, artificially induced, or recombinant, which has specific immunoreactive activity. Normally, as used herein, an antibody will include at least one variable region from a heavy or light chain. Additionally, the antibody may comprise combinations of variable regions. The combination may include more than one variable region of a light chain or of a heavy chain. The antibody may also include variable regions from one or more light chains in combination with variable regions of one or more heavy chains.

Accordingly, a fragment of a naturally occurring or recombinant antibody molecule is encompassed within the scope of this invention. As used herein a Fab protein of a F(ab′)2 protein which exhibits immunoreactive activity is an antibody. Also, as used herein, an immunologically reactive complex comprising two different polypeptides, the shorter of which function as a light chain and the longer of which function as a heavy chain is an antibody. Further, as used herein, a variable region fragment (Fv region) is also an antibody.

Further, in one example, the immunologically reactive complex may be produced by known genetic engineering methods or otherwise (4, 8, 9, 5, 10).

In another example, the previously described anti-idiotypic antibody, preferably monoclonal antibody, which specifically induces an immune response against the GD₃ ganglioside also specifically binds to the binding site of the R24 antibody.

The genetically engineered anti-idiotypic antibody of this invention may be derived from any mammal. The genetically engineered anti-idiotypic antibody may also be a chimeric antibody derived from a combination of different mammals. The mammal may be, for example, a rabbit, a mouse, a rat, a goat, or a human. The combination of different mammals includes fragments from human and mouse sources.

The antibody class and sub-class is not critical. Any sub-class may be used to prepare the anti-idiotypic antibodies of the invention. The antibodies may also contain fragments from antibodies of different classes and sub-classes, thereby forming a composite. In one preferred embodiment of the invention, the above-described anti-idiotypic antibody is an IgG class anti-idiotypic antibody.

In one example, the previously-described anti-idiotypic antibody, preferably monoclonal, is designated BEC2. The present invention also provides a hybridoma which produces any of the above-described anti-idiotypic antibodies. The hybridoma that produces BEC2 is designated BEC2 hybridoma. The BEC2 hybridoma has been deposited pursuant to the Budapest Treaty On the International Recognition Of The Deposit of Microorganisms For The Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. HB 10153. The present invention further provides an anti-idiotypic antibody produced by the BEC2 hybridoma.

In one embodiment of this invention, the anti-idiotypic antibody is labeled with a detectable marker. The detectable marker may be created by an enzyme reaction, for example, alkaline-phosphatase or horseradish peroxidase.

It would be clear to those skilled in the art that one method of attaching the subject anti-idiotypic antibody to an enzyme, would be by a modification of the periodate method (6). Alternatively, it would be clear to those skilled in the art to attach a ligand to the subject anti-idiotypic antibodies and a receptor of the ligand to the enzyme. A suitable ligand receptor combination is biotin-avidin in accordance with the method of Bayer et al. (7).

Moreover, this invention provides a method of generating the above-described anti-idiotypic antibody, for example BEC2, which comprises: (a) preparing an antibody, preferably a monoclonal antibody, against a glycosphingolipid, more specifically against a ganglioside, and more specifically against GD₃; (b) purifying the antibody; (c) attaching the antibody, such as R24, to a carrier so as to produce an immunogen; (d) combining the immunogen with a cytokine so as to produce a novel immunogen-cytokine combination; and (e) injecting the novel immunogen-cytokine combination into a mammal thereby producing the anti-idiotypic antibody.

In accordance with the practice of the invention the carrier may be any material which when combined with the antibody is immunogenic. The carrier may be a microbe, a liposome, or a proteosome. In one example the microbe is S. aureus.

Additionally, in accordance with the practice of the invention the cytokine may be an interleukin, preferably interleukin l. In one example, interleukin l is recombinant human interleukin l.

GD₃ is poorly immunogenic in man. In contrast, the subject anti-idiotypic antibodies are strongly immunogenic.

The present invention provides vaccine comprising any of the anti-idiotypic antibodies, in particular BEC2, and a pharmaceutically acceptable carrier. These vaccines are highly potent and commercially useful therapeutics.

Examples of suitable carriers are well known in the art and may include, but are in no way and are not intended to be limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.

Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium of calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods.

Such carriers are well known in the art and may include, but are in no way and are not intended to be limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.

Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods.

This invention further provides a method of immunizing a human subject against GD₃ gangliosides. The method comprises administering to the subject a suitable dose of the above-described vaccine. Administration of any of the vaccines may be effected by various ways. Suitable means of administration includes intravenous, intraperitoneal, subcutaneous, or intramuscular, topical, or intradermal administration.

Additionally, this invention provides a method of treating a human subject with a neoplastic or preneoplastic condition associated with the expression of glycosphingolipids, for example gangliosides such as GD3. The method comprises administering to the subject an effective amount of any of the previously-described anti-idiotypic antibodies and a pharmaceutically acceptable carrier. Examples of neoplastic and preneoplastic conditions associated with expression of GD₃ gangliosides include a condition selected from a group consisting of a melanoma, squamous cell carcinoma, glioblastoma, sarcoma, T cell leukemia and lymphoma, Hodgkin's disease, small cell carcinoma of the lung, or brain tumor.

In one example, administration of the previously-described anti-idiotypic antibody is effected by intravenous administration. In another example of the invention, administration of the previously-described anti-idiotypic antibody is effected by intraperitoneal administration. In yet another example, administration of the previously-described anti-idiotypic antibody is effected by subcutaneous administration. In still another example of the invention, administration of the previously-described anti-idiotypic antibody is effected by intramuscular administration. Further, in another example of the invention, administration of the previously-described anti-idiotypic antibody is effected by topical administration. Additionally, in yet another example, administration of the previously-described anti-idiotypic antibody is effected by intradermal administration.

This invention also provides a method of inhibiting a microbial infection, such as an endotoxin-related infection, in a subject which comprises priming the subject with an effective amount of the previously-described vaccine in order to generate an antibody which specifically binds to a glycosphingolipid on a microbial membrane, the antibody and the glycosphingolipid forming a combination and thereby inhibiting the microbial infection.

Additionally, this invention provides a method for inhibiting a malignancy associated with expression of GD₃ gangliosides in a subject such as a human subject. The method comprises priming the subject with an effective amount of the previously-described vaccine in order to generate an antibody which specifically binds to a GD₃ ganglioside, the antibody and the GD₃ ganglioside forming a combination and thereby inhibiting a malignancy associated with expression of GD₃ gangliosides. Examples of malignancies associated with expression of GD₃ gangliosides include a malignancy selected from a group consisting of a melanoma, squamous cell carcinoma, glioblastoma, sarcoma, T cell leukemia and lymphoma, Hodgkin's disease, small cell carcinoma of the lung, or brain tumor.

In one example, the method for measuring expression levels of GD₃ gangliosides in tissue from a subject is effected by determining the amount and distribution of GD₃ in tissue sections from a neoplastic tissue to be tested. The method comprises contacting the tissue to be tested with the R24 antibody and thereby detecting the expression level and distribution of GD₃.

The present invention provides a method of treating a subject suffering from a malignancy associated with expression of GD₃ gangliosides on malignant cells. The method comprises administering to the subject a cell killing amount of an anti-GD3 antibody labeled with a cytotoxic agent, such as a radioisotope, a drug, or a heavy metal. The labeled antibody binds to GD3 ganglioside, after which the cytotoxic agent kills the malignant cells to which the GD3 ganglioside is attached. It is important to eliminate unbound antibody labeled with a cytotoxic agent quickly. Elimination may be expedited by contacting the unbound labeled antibody with any of the previously-described anti-idiotypic antibodies. The anti-idiotypic antibodies bind the previously unbound labeled antibody so as to form a complex which may be cleared by excretion from the subject. In this way the subject is treated for the malignancy without undue harm by the cytotoxic agent.

It would be well known to those skilled in the art that “clearing” undesirable composites, in particular the labeled complex, would involve transport of the complex to the spleen, lung, or liver, wherein an Fc region of the antibody within the composite would bind to the Fc receptor of spleen, lung, or liver tissues or cells derived therefrom. After binding to these tissues or the cells derived therefrom, the complex is excreted from the body by physiological or biochemical means.

Examples of a malignancy associated with the expression of GD₃ gangliosides includes any malignancy selected from a group consisting of a melanoma, squamous cell carcinoma, glioblastoma, sarcoma, T cell leukemia and lymphoma, Hodgkin's disease, small cell carcinoma of the lung, or brain tumor.

This invention additionally provides a method of inhibiting the proliferation of cells associated with elevated levels of GD₃ gangliosides in a subject. The method comprises administering to the subject an effective amount of the previously-described vaccine.

Further, this invention provides an immunogenic composition of matter comprising an effective amount of a cytokine, e.g. interleukin l, preferably recombinant human interleukin l, and a melanoma ganglioside-specific antibody, such as a R24 antibody, attached to a carrier. Further, the carrier may be a microbe, preferably S. aureus.

Additionally, the present invention provides a method of generating an anti-idiotypic antibody which comprises: (a) attaching an antigen, for example R24, to a microbe thereby obtaining an immunogen; (b) purifying the immunogen; (c) combining the immunogen with interleukin l so as to produce an immunogen-interleukin l combination; and (d) injecting the immunogen-interleukin l combination into mice, preferably syngeneic mice, thereby generating the anti-idiotypic antibody.

Finally, this invention is illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow.

Experimental Details

Anti-idiotypic mAbs to R24

We immunized syngeneic (C57B1/6X BALB/C)F1 female mice with purified R24 free of variants V1-R24 and V2-R24. The significance of using syngeneic mice is that the only portion of the R24 molecule which should be recognized as foreign is the paratope. It is standard to mix the immunizing material with an “adjuvant”, a potentiator of an immune response, which boosts the immune response of the mouse. The novel adjuvant disclosed herein was made by sticking the R24 onto S. aureus cells and injecting the R24-coated cells along with rh IL-1 (DuPont).

The mice received two weekly boosts. Three days before fusion, mice were injected with 50 μg R24 i.v. Splenocytes were fused with SP2/0 using standard techniques. Hybridomas were screened for the ability to bind to R24 F(ab′)₂ fragments using an alkaline phosphatase conjugated goat anti-mouse F_(c)-specific second antibody. Positive colonies were subcloned twice by limiting dilution. Both S. aureus and IL-1 are strong stimulators of B lymphocytes. Using standard hybridoma techniques and fusion of immune mouse spleen cells to the SP2/0 mouse myeloma cell line BEC2 and BEC3 were produced.

Demonstrating that BEC2 is an Anti-idiotypic mAb

BEC2 recognizes unique determinants on R24

If BEC2 is indeed an anti-idiotype for R24, it would be expected that BEC2 would bind only to R24 and no other mAb. We have tested the ability of BEC2 to bind to 23 other mAbs using an inhibition assay. No mAb, other than R24, can compete with R24 for BEC2 binding (Table 1). It is notable that V1-R24, the variant with two irrelevant light chains, is not bound by BEC2. This is consistent with the model that BEC2 recognizes the paratope of R24.

The results in Table 1 were obtained using the following method. BEC2 (5 μg/ml) was pre-incubated with dilutions of inhibiting mAb for 1 hr. The mixture was then, plated into wells previously coated with R24 F(ab′)₂ fragments and blocked with 5% non-fat milk. BEC2 binding was detected by ELISA using an alkaline phosphatase-conjugated second antibody specific for the F_(c) region. BEC2 carries the internal image of the original antigen GD₃

An anti-idiotypic mAb which recognizes the paratope of the immunizing mAb is called an Ab2b anti-idiotypic mAb. One implication of this is that the paratope of BEC2 should mimic GD₃. If one imagines the R24 paratope as a template for GD₃, and both GD₃ and BEC2 fit that template, then BEC2 must resemble GD₃. Using serological assays, we show that BEC2 can inhibit the binding of R24 to GD₃. FIG. 4 shows the results on one experiment in which different concentrations of BEC2 were preincubated with a fixed dilution of biotinylated R24. The mixture was added to a well coated with GD₃ and the amount of R24 available for binding was measured by adding alkaline phosphatase-conjugated avidin. The data show significant inhibition of R24 binding to GD₃ by BEC2 which is consistent with BEC2 being an Ab2b anti-idiotypic mAb. In order to directly demonstrate that the paratope of BEC2 mimics GD₃, it is necessary to immunize animals with BEC2 and show that the animals develop antibodies to GD₃.

We immunized four New Zealand White Rabbits with BEC2 mixed with Freund's complete adjuvant. The animals received several booster immunizations mixed with Freund's incomplete adjuvant

TABLE 1 Ability of BEC2 to bind to various monoclonal antibodies as measured by the ability of the monoclonal antibody to prevent binding of BEC2 to R24. Inhibition of BEC2 binding to R24 F(ab′)₂ Fragments by a Panel of mAb. Antibody Isotype Specificity IC501  1. MOPC104E M &1—>3 dextrans 390  2. TEPC183 M ND2 425  3. MOPC315 A Dinitrophenol . . . >900  4. TEPC15 A Phosphoryl choline >900  5. MOPC21 G1 ND >700  6. HT29-15 G1 Adenocarcinoma >900  7. 455 G1 EGF receptor >900  8. M111 G1 gp110 465  9. S22 G1 ND >1000 10. 2G10 G1 gp76 >1000 11. C350 G1 gp180 >500 12. UPC10 G2a b2-6 fructosan >900 13. TA99 G2a gp75 >900 14. A33 G2a ND >900 15. M195 G2a CD33 >1000 16. F23 G2a CD13 >1000 17. MOPC141 G2b ND 485 18. OKB7 G2b CD21 >1000 19. FLOPC21 G3 ND >900 20. Y5606 G3 AMP/purine/tri- >500 ethanolamine 21. F36/22 G3 Breast Carcinoma >1000 22. 3F8 G3 GD2 ganglioside >1000 23. V1-R243 G3 GD₃ ganglioside 425 24. R24 G3 GD₃ ganglioside 26 ¹IC₅₀ is the concentration of inhibiting mAb(in 1 g/ml) required to inhibit 50% of BEC2 binding to R24 F(ab′)₂ fragments. ²Not Determined ³A variant of R24 in which both light chains are replaced with MOPC 21 myeloma light chains. This results in a mAb with >40-fold lower affinity for GD₃.

over approximately three months. The rabbits were bled periodically and the serum was assayed for ant-GD₃ reactivity using two different assays. The anti-GD₃ IgG response was measured by radioimmunoassay (RIA) using [¹²⁵I]-Protein A. The anti-GD₃ IgM response was measured by ELISA using alkaline phosphatase-conjugated goat anti-rabbit second antibody which was specific for the mu chain. The specificity of both the IgG and IgM response was analyzed by immunoblot assays against several gangliosides. In these assays, [¹²⁵I]-Protein A was used to visualize IgG binding while peroxidase-conjugated goat anti-rabbit (mu chain specific) antiserum was used to visualize IgM binding.

At least three of the four rabbits developed an anti-GD₃ antibody response. By RIA, Rabbit 521 developed IgG against GD₃ (FIG. 5) although by immunoblotting, all four rabbits appeared to make anti-GD₃ IgG. By ELISA, rabbits 543 (FIG. 6) and 545 (FIG. 7) developed IgM. The specificity of this antibody response was analyzed by immunoblot. FIG. 8 shows that IgM from rabbit 543 binds to GD₃ but not to the other five gangliosides tested (GM1, GM2, GM3, GD1a, GT1b). As noted above, immunoblotting of serum from all four rabbits showed evidence of IgG binding to GD₃ and this binding was specific.

In order to confirm that this anti-GD₃ reactivity was indeed IgG, serum from rabbit 543 was precipitated using 45% NH₄SO₄ and the immunoglobulin salted out was fractionated by size exclusion chromatography (Superose 6 column, Pharmacia). FIG. 9 shows that most of the protein eluted at a molecular weight of approximately 120 kD which is consistent with IgG (145-150 kD) but not consistent with IgM (900 kD). FIG. 10 shows the anti-GD₃ reactivity of each fraction and it is clear that only the 120 kD fraction bind to GD₃.

Results

BEC2 binds to the Fab region of R24 and blocks R24 binding to GD₃ gangliosides. Additionally, BEC2 binds to two anti-GD₃ monoclonal antibodies. C5(IgG3) and K9 (IgM). Because BEC2 contains irrelevant light chains BEC2 neither binds to other mouse monoclonal antibodies, including four IgG3 monoclonal antibodies, nor binds to the variant of R24.

BEC2 is an AB2 anti-idiotypic monoclonal antibody for it binds to the portion of R24 which recognizes GD₃. If one imagines the R24 binding site as a template for GD₃, and both GD₃ and BEC2 fit that template, then BEC2 must resemble GD₃. Using serological assays, we have shown that BEC2 does indeed resemble GD₃. BEC2 is a useful way to immunize against GD₃, and thus, important as a vaccine against melanoma and other tumors.

To demonstrate that BEC2 carries an internal image of GD₃, we immunized NZW rabbits with BEC2 and studied pre-immune and immune sera for evidence of anti-GD₃ reactivity. Immunized rabbits developed an antibody response to GD₃ detectable by ELISA and Immunoblot against purified GD₃, and by mixed hemadsorption assay against a GD₃+ human melanoma cell line. Specificity analysis showed that immune sera recognized only GD₃ and not GM₁, GM₂, GM₃, GD_(1a), or GT_(1b), monoclonal antibody BEC2 provides a strategy for active immunization of melanoma patients against GD₃.

Immunization with BEC2 induces high titer, specific IgM and IgG antibodies to GD₃ in rabbits and IgM antibodies in syngeneic (BALB/c×C57B1)F1 mice.

From the experiments, we find that BEC2 is a mouse IgG2b anti-idiotypic mAb directed against R24 and binds the antigen-binding site of R24. Further, immunization of rabbits with BEC2 results in an IgM and IgG response against GD₃. Finally, BEC2 carries the internal image of the ganglioside GD₃, therefore, it is useful for immunization against GD₃ gangliosides.

REFERENCES

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5. Morrison, S. L. et al. (1987) Ann. N.Y. Acad. Sci. 507:187.

6. Oi, V. T., and Morrison, S. L. (1986) BioTechniques 4:214.

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What is claimed is:
 1. A method for generating in a subject an immune response against ganglioside GD₃ by administering to the subject anti-idiotypic antibody BEC-2 (ATCC No. HB 10153). 